Role of dysregulated miRNA in Tregs in Multiple Sclerosis

Tregs 中失调的 miRNA 在多发性硬化症中的作用

• 批准号: 8463637
• 负责人: Amy E Lovett-Racke
• 金额: $ 18.4万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-10-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8463637
• 关键词: 3' Untranslated Regions; Affect; Algorithms; Autoimmune Diseases; Biological Assay; CD4 Positive T Lymphocytes; Cell Differentiation process; Cells; Characteristics; Defect; Development; Disease; Disease Progression; Disease susceptibility; Environment; Equilibrium; Figs - dietary; Functional disorder; Gene Expression; Gene Targeting; Genes; Health; Human; Immune; Immune system; Inflammatory; Laboratories; Luciferases; Maintenance; Mediating; MicroRNAs; Multiple Sclerosis; Nerve Degeneration; Neurologic; Pathology; Pathway interactions; Patients; Phenotype; Play; Predisposition; Production; Proteins; RNA Interference Pathway; Regulatory T-Lymphocyte; Relative (related person); Role; Signal Pathway; Signal Transduction; Sorting - Cell Movement; T-Lymphocyte; Th1 Cells; Th2 Cells; Work; central nervous system demyelinating disorder; cytokine; disability; in vivo; inhibitor/antagonist; innovation; memory CD4 T lymphocyte; novel; peripheral blood; receptor; young adult

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) which can result in severe neurological deficits. The cause of the disease is unknown, the immune and neurodegenerative mechanisms underlying the pathophysiology of the disease are poorly understood, and current therapies are only partially effective at slowing disease progression. My laboratory has performed a large miRNA profiling study on naive and effector/memory CD4 T cell in untreated MS patients to determine if miRNA may contribute to disease susceptibility or progression. The TGF¿ signaling pathway was predicted to be suppressed in MS patients by 12 miRNAs identified as being dysregulated in CD4 T cells. TGF¿ has shown to play a vital role in CD4+CD25hiFoxp3+ regulatory T cells (Tregs), and Tregs have been shown to be defective in MS patients. TGF¿ signaling has been shown to be critical for production of iTregs, and the maintenance of suppressor functions and Foxp3 expression in Tregs. We hypothesize that dysregulated miRNA expression in CD4 T cells in MS patients' results in impaired TGF¿ signaling and defective CD4 regulatory T cells, promoting the onset and progression of MS. Aim 1: To determine if miRNAs identified as dysregulated in CD4 T cells in MS patients directly target specific molecules in the TGF ¿ signaling pathway. First, we will determine if there is a correlation in gene expression levels between specific miRNAs and predicted TGF¿ targets in human CD4 T cells from MS patients and healthy controls. Second, we will use luciferase assays to determine if specific miRNAs suppress the 3' UTR sequence of the predicted target genes in the TGF¿ pathway. Aim 2: To determine if the miRNAs that target the TGF¿ pathway alter iTreg development and/or suppressive functions of Tregs. TGF¿ targeting miRNAs will be inhibited or over-expressed in naive CD4 T cells and then cultured under iTreg conditions. The resulting T cells will be phenotyped for iTreg characteristics. In addition, Tregs will be transfected with specific miRNA inhibitors, evaluated for sensitivity to TGF¿, and ability to suppress effector T cells. Aim 3: To determine if there is an association between the level of TGF¿-associated miRNA expression and the number of Tregs in vivo, or an association between TGF¿-associated miRNA expression and suppressive capacity of Tregs in MS patients. Flow cytometric analysis of peripheral blood CD4 T cells from MS patients will evaluate the number of Tregs in MS patients relative to the expression of TGF¿-associated miRNAs. In addition, Tregs will be sorted from peripheral blood of MS patients, evaluated for suppressive capacity, and determine if there is a correlation between TGF¿-associated miRNAs and Treg number and/or function. Given the well-documented role of Tregs in maintaining tolerance to self and the observation that CD4 Tregs are defective in MS patients, this novel study will determine if dysregulated miRNA expression in CD4 T cells underlies this Treg defect in MS patients. Although several studies have identified miRNAs that play a role in Tregs17-22, no studies have identified differentially expressed miRNAs in MS patients that are specifically associated with genes in the TG¿ pathway and their influence on Tregs in an autoimmune disease. Treg cells play an important role in the delicate balance of protection from our environment and protecting ourselves from our own immune system, and TGF¿ has pleiotrophic effects that extend beyond Tregs that have vast importance in health and disease, making this an invaluable study.

描述（由申请人提供）：多发性硬化症（MS）是一种免疫介导的中枢神经系统（CNS）脱髓鞘疾病，可导致严重的神经缺陷。该疾病的病因尚不清楚，对该疾病病理生理学背后的免疫和神经退行性机制知之甚少，并且目前的疗法在减缓疾病进展方面仅部分有效。我的实验室对未经治疗的多发性硬化症患者的幼稚和效应/记忆 CD4 T 细胞进行了一项大型 miRNA 分析研究，以确定 miRNA 是否可能导致疾病易感性或进展。预计多发性硬化症患者中的 TGF¿ 信号通路会受到 CD4 T 细胞中被鉴定为失调的 12 种 miRNA 的抑制。 TGF¿ 已被证明在 CD4+CD25hiFoxp3+ 调节性 T 细胞 (Treg) 中发挥着至关重要的作用，而 Tregs 已被证明在多发性硬化症患者中存在缺陷。 TGF¿ 信号传导已被证明对于 iTreg 的产生以及 Tregs 中抑制功能和 Foxp3 表达的维持至关重要。我们假设多发性硬化症患者的 CD4 T 细胞中 miRNA 表达失调会导致 TGFβ 信号传导受损和 CD4 调节性 T 细胞有缺陷，从而促进多发性硬化症的发病和进展。 目标 1：确定 MS 患者 CD4 T 细胞中被鉴定为失调的 miRNA 是否直接靶向 TGF ¿ 信号通路中的特定分子。首先，我们将确定来自多发性硬化症患者和健康对照的人类 CD4 T 细胞中特定 miRNA 与预测的 TGF 靶点之间的基因表达水平是否存在相关性。其次，我们将使用荧光素酶测定来确定特定 miRNA 是否抑制 TGF¿ 途径中预测靶基因的 3' UTR 序列。 目标 2：确定靶向 TGF¿ 通路的 miRNA 是否会改变 iTreg 的发育和/或 Tregs 的抑制功能。 TGF¿ 靶向 miRNA 将在初始 CD4 T 细胞中被抑制或过度表达，然后在 iTreg 条件下培养。所得 T 细胞将针对 iTreg 特征进行表型分析。此外，Tregs 将被特定的 miRNA 抑制剂转染，评估其对 TGF 的敏感性以及抑制效应 T 细胞的能力。 目标 3：确定多发性硬化症患者体内 TGFβ 相关 miRNA 表达水平与 Tregs 数量之间是否存在关联，或者 TGFβ 相关 miRNA 表达与 Tregs 抑制能力之间是否存在关联。对多发性硬化症患者外周血 CD4 T 细胞进行流式细胞术分析，将评估多发性硬化症患者中与 TGF 相关 miRNA 表达相关的 Tregs 数量。此外，将从多发性硬化症患者的外周血中分选Tregs，评估其抑制能力，并确定TGFβ相关miRNA与Tregs数量和/或功能之间是否存在相关性。 鉴于Tregs在维持自我耐受性方面的作用已得到充分证明，并且观察到CD4 Tregs在MS患者中存在缺陷，这项新研究将确定CD4 T细胞中的miRNA表达失调是否是MS患者中这种Treg缺陷的基础。尽管一些研究已经鉴定出在 Tregs17-22 中发挥作用的 miRNA，但没有研究鉴定出 MS 患者中差异表达的 miRNA 与 TG¿ 通路中的基因特异性相关，以及它们对自身免疫性疾病中的 Tregs 的影响。 Treg 细胞在保护我们免受环境侵害和保护我们自己免受自身免疫系统侵害的微妙平衡中发挥着重要作用，而 TGF 的多效作用超出了在健康和疾病中具有巨大重要性的 Tregs，这使得这项研究变得非常有价值。